Specific Transcription Factor Binding Sites Research Articles

Integrative computational analyses implicate regulatory genomic elements contributing to spina bifida

PurposeSpina bifida (SB) arises from complex genetic interactions that converge to interfere with neural tube closure. Understanding the precise patterns conferring SB risk requires a deep exploration of the genomic networks and molecular pathways that govern neurulation. This study aims to delineate genome-wide regulatory signatures underlying SB pathophysiology, MethodsAn untargeted, genome-wide approach was used to interrogate regulatory regions for rare single-nucleotide and copy-number variants (rSNVs and rCNVs, respectively) predicted to affect gene expression, comparing results from SB patients with healthy controls. Qualifying variants were subjected to a deep learning prioritization framework to identify the most functionally relevant variants, as well as the likely target genes affected by these rare regulatory variants. ResultsThis ensemble of computational tools identified rSNVs in specific transcription factor binding sites (TFBSs) that distinguish SB cases from controls. rSNV enrichment was found in specific TFBSs, especially CCCTC-binding factor binding sites. These TFBSs were subjected to a deep learning prioritization framework to identify the most functionally relevant variants, as well as the likely target genes affected by these rSNVs. The functional pathways or modules implicated by these regulated genes serve protein transport, cilia assembly, and central nervous system development. Moreover, the detected rare copy-number variants in SB cases are positioned to disrupt gene regulatory networks and alter 3-dimensional genomic architectures, including brain-specific enhancers and topologically associated domain boundaries of relevant cell types. ConclusionOur study provides a resource for identifying and interpreting genomic regulatory DNA variant contributions to human SB genetic predisposition.

Chromatin Accessibility Profiling and Data Analysis Using ATAC-seq in Nothobranchius furzeri.

The state of genome-wide chromatin accessibility in cells, tissues, or organisms can be investigated with a technique called assay for transposase-accessible chromatin using sequencing (ATAC-seq). ATAC-seq is a powerful approach for profiling the epigenomic landscape of cells using very low input materials. Analysis of chromatin accessibility data allows for prediction of gene expression and identification of regulatory elements such as potential enhancers and specific transcription-factor binding sites. Here, we describe an optimized ATAC-seq protocol for the preparation of isolated nuclei and subsequent next-generation sequencing from whole embryos and tissues of the African turquoise killifish (Nothobranchius furzeri). Importantly, we provide an overview of a pipeline for processing and analyzing ATAC-seq data from killifish.

Oxytocin variation and brain region-specific gene expression in a domesticated avian species.

The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia (WRM). Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to WRMs. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We sequenced the OT from 10 wild munias and 11 Bengalese finches and identified intra-strain variability in both the untranslated and protein-coding regions of the sequence, with all the latter giving rise to synonymous mutations. Several of these changes fall in specific transcription factor-binding sites, and show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed a significantly higher OT mRNA expression in the cerebrum of the Bengalese finches relative to munias, but a significantly lower expression in their diencephalon. Our study thus points to a brain region-specific pattern of neurochemical expression in domesticated and wild avian strains, which could be linked to domestication and the behavioral changes associated with it.

A flexible repertoire of transcription factor binding sites and a diversity threshold determines enhancer activity in embryonic stem cells

Transcriptional enhancers are critical for development and phenotype evolution and are often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. To examine the enhancer regulatory code for pluripotent stem cells, we identified genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs). Examination of these regions revealed that they contain on average 12.6 conserved transcription factor binding site (TFBS) sequences. Enriched TFBSs are a diverse repertoire of 70 different sequences representing the binding sequences of both known and novel ESC regulators. Using a diverse set of TFBSs from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site-directed mutagenesis of conserved TFBSs in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for 10 or more different TFBSs. Furthermore, specific TFBSs, including the POU5F1:SOX2 comotif, are dispensable, despite cobinding the POU5F1 (also known as OCT4), SOX2, and NANOG master regulators of pluripotency. These findings reveal that a TFBS sequence diversity threshold overrides the need for optimized regulatory grammar and individual TFBSs that recruit specific master regulators.

Unexplored regulatory sequences of divergently paired GLA and HNRNPH2 loci pertinent to Fabry disease in human kidney and skin cells: Presence of an active bidirectional promoter.

Fabry disease (FD) is a rare hereditary disorder characterized by a wide range of symptoms caused by a variety of mutations in the galactosidase α (GLA) gene. The heterogeneous nuclear ribonucleoprotein (HNRNPH2) gene is divergently paired with GLA on chromosome X and is thought to be implicated in FD. However, insufficient information is available on the regulatory mechanisms associated with the expression of HNRNPH2 and the GLA loci. Therefore, the current study performed bioinformatics analyses to assess the GLA and HNRNPH2 loci and investigate the regulatory mechanisms involved in the expression of each gene. The regulatory mechanisms underlying GLA and HNRNPH2 were revealed. The expression of each gene was associated with a bidirectional promoter (BDP) characterized by the absence of TATA box motifs and the presence of specific transcription factor binding sites (TFBSs) and a CpG Island (CGI). The nuclear run-on transcription assay confirmed the activity of BDP GLA and HNRNPH2 transcription in 293T. Methylation-specific PCR analysis demonstrated a statistically significant variation in the DNA methylation pattern of BDP in several cell lines, including human adult epidermal keratinocytes (AEKs), human renal glomerular endothelial cells, human renal epithelial cells and 293T cells. The highest observed significance was demonstrated in AEKs (P<0.05). The results of the chromatin-immunoprecipitation assay using 293T cells identified specific TFBS motifs for Yin Yang 1 and nuclear respiratory factor 1 transcription factors in BDPs. The National Center for Biotechnology Information-single nucleotide polymorphism database revealed pathogenic variants in the BDP sequence. Additionally, a previously reported variant associated with a severe heterozygous female case of GLA FD was mapped in BDP. The results of the present study suggested that the expression of the divergent paired loci, GLA and HNRNPH2, were controlled by BDP. Mutations in BDP may also serve a role in FD and may explain clinical disease diversity.

The human olfactory transcriptome.

BackgroundOlfaction is a versatile sensory mechanism for detecting thousands of volatile odorants. Although molecular basis of odorant signaling is relatively well understood considerable gaps remain in the complete charting of all relevant gene products. To address this challenge, we applied RNAseq to four well-characterized human olfactory epithelial samples and compared the results to novel and published mouse olfactory epithelium as well as 16 human control tissues.ResultsWe identified 194 non-olfactory receptor (OR) genes that are overexpressed in human olfactory tissues vs. controls. The highest overexpression is seen for lipocalins and bactericidal/permeability-increasing (BPI)-fold proteins, which in other species include secreted odorant carriers. Mouse-human discordance in orthologous lipocalin expression suggests different mammalian evolutionary paths in this family.Of the overexpressed genes 36 have documented olfactory function while for 158 there is little or no previous such functional evidence. The latter group includes GPCRs, neuropeptides, solute carriers, transcription factors and biotransformation enzymes. Many of them may be indirectly implicated in sensory function, and ~70 % are over expressed also in mouse olfactory epithelium, corroborating their olfactory role.Nearly 90 % of the intact OR repertoire, and ~60 % of the OR pseudogenes are expressed in the olfactory epithelium, with the latter showing a 3-fold lower expression. ORs transcription levels show a 1000-fold inter-paralog variation, as well as significant inter-individual differences. We assembled 160 transcripts representing 100 intact OR genes. These include 1–4 short 5’ non-coding exons with considerable alternative splicing and long last exons that contain the coding region and 3’ untranslated region of highly variable length. Notably, we identified 10 ORs with an intact open reading frame but with seemingly non-functional transcripts, suggesting a yet unreported OR pseudogenization mechanism. Analysis of the OR upstream regions indicated an enrichment of the homeobox family transcription factor binding sites and a consensus localization of a specific transcription factor binding site subfamily (Olf/EBF).ConclusionsWe provide an overview of expression levels of ORs and auxiliary genes in human olfactory epithelium. This forms a transcriptomic view of the entire OR repertoire, and reveals a large number of over-expressed uncharacterized human non-receptor genes, providing a platform for future discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2960-3) contains supplementary material, which is available to authorized users.

Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using in vitro TF-DNA preferences obtained from the universal protein binding microarrays (PBM) for ~90 eukaryotic TFs belonging to 22 different DNA-binding domain types. As a result of this new analysis, we conclude that nonconsensus protein-DNA binding is a widespread phenomenon that significantly affects protein-DNA binding preferences and need not require the presence of consensus (specific) TFBSs in order to achieve genome-wide TF-DNA binding specificity.

Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.

In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group 3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing group 1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group 2 and 3 viruses showed intermediate or no replication, respectively. A literature search revealed unnoticed group 1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease. The NCCRs of polyomaviruses mediate silent persistence of the viral genome as well as the appropriately timed (re)activation of the viral life cycle. This study indicates that the basal BKPyV NCCR is critically controlled by a hierarchy of single TFBS in the archetype NCCR that direct, modulate, and execute the bidirectional early and late viral gene expression. The results provide new insights into how BKPyV NCCR functions as a viral sensor of host cell signals and shed new light on how transcription factors like Sp1 control bidirectional viral gene expression and contribute to replication and pathology.

Protein-DNA Binding in the Absence of Consensus Binding Motif

Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)-DNA binding. In our study we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF-DNA binding preferences. We used high-throughput protein-DNA binding assays to measure the binding levels for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein-DNA binding mechanism induced by DNA sequence symmetry in the absence of consensus binding motif, and experimentally demonstrate that this mechanism indeed highly affects protein-DNA binding preferences.

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin.

The development of cisplatin resistance in human cancers is controlled by multiple genes and leads to therapeutic failure. Hypermethylation of specific gene promoters is a key event in clinical resistance to cisplatin. Although the usage of multiple promoters is frequent in the transcription of human genes, the role of alternative promoters and their regulatory sequences have not yet been investigated in cisplatin resistance genes. In a new approach, we hypothesized that human cancers exploit the specific transcription factor-binding sites (TFBS) and CpG islands (CGIs) located in the alternative promoters of certain genes to acquire platinum drug resistance. To provide a useful resource of regulatory elements associated with cisplatin resistance, we investigated the TFBS and CGIs in 48 alternative promoters of 14 hypermethylated cisplatin resistance genes previously reported. CGIs prone to methylation were identified in 28 alternative promoters of 11 hypermethylated genes. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, whereas the ones lacking CGIs were distributed in two unrelated subclasses. Regulatory sequences, initiator and TATA-532 prevailed over TATA-8 and were found in all the promoters. B recognition element (BRE) sequences were present only in alternative promoters harboring CGIs, but CCAAT and TAACC were found in both types of alternative promoters, whereas downstream promoter element sequences were significantly less frequent. Therefore, it was hypothesized that BRE and CGI sequences co-localized in alternative promoters of cisplatin resistance genes may be used to design molecular markers for drug resistance. A more extensive knowledge of alternative promoters and their regulatory elements in clinical resistance to cisplatin is likely to usher novel avenues for sensitizing human cancers to treatment.

A Conserved Apomixis-Specific Polymorphism Is Correlated with Exclusive Exonuclease Expression in Premeiotic Ovules of Apomictic Boechera Species

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by its parthenogenetic development into offspring that are genetic clones of the mother plant. Fertilization (i.e. pseudogamy) of the central cell is important for the production of a functional endosperm with a balanced 2:1 maternal:paternal genome ratio. Here, we present the APOLLO (for apomixis-linked locus) gene, an Aspartate Glutamate Aspartate Aspartate histidine exonuclease whose transcripts are down-regulated in sexual ovules entering meiosis while being up-regulated in apomeiotic ovules at the same stage of development in plants of the genus Boechera. APOLLO has both "apoalleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sexalleles." All apomictic Boechera spp. accessions proved to be heterozygous for the APOLLO gene (having at least one apoallele and one sexallele), while all sexual genotypes were homozygous for sexalleles. Apoalleles contained a 20-nucleotide polymorphism present in the 5' untranslated region that contains specific transcription factor-binding sites for ARABIDOPSIS THALIANA HOMEOBOX PROTEIN5, LIM1 (for LINEAGE ABNORMAL11, INSULIN1, MECHANOSENSORY PROTEIN3), SORLIP1AT (for SEQUENCES OVERREPRESENTED IN LIGHT-INDUCED PROMOTERS IN ARABIDOPSIS THALIANA1), SORLIP2AT, and POLYA SIGNAL1. In the same region, sexalleles contain transcription factor-binding sites for DNA BINDING WITH ONE FINGER2, DNA BINDING WITH ONE FINGER3, and PROLAMIN BOX-BINDING FACTOR. Our results suggest that the expression of a single deregulated allele could induce the cascade of events leading to asexual female gamete formation in an apomictic plant.

Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model

Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ∼5,000 synthetic regulatory elements containing patterns from 12 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.

Aldosterone-dependenttrans-activation and epigenetic derepression of ENaC: where is the balance?

the critical role of the kidney collecting duct and the epithelial Na+ channel (ENaC) in the regulation of arterial blood pressure has been recognized for many years (5). However, the factors that alter renal function and the pathways involved remain to be determined. The mineralocorticoid hormone aldosterone increases tubular sodium absorption in large part by increasing α-ENaC transcription. The pioneering demonstration of epigenetic regulation of ENaC was published by the Kone's laboratory several years ago, when it was reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the α-ENaC promoter and that aldosterone and serum- and glucocorticoid-induced kinase-1 (Sgk1) act to disrupt this complex and its inhibitory effects (8, 9). Following studies indicated that either Af9 (in humans, encoded by MLLT3) or Sirt1 (encoded by SIRT1) directly binds to +78/+92 of α-ENaC and recruits Dot1a to repress basal and aldosterone-sensitive α-ENaC transcription (7, 10). Despite this epigenetic repression, basal α-ENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In an issue of the American Journal of Physiology-Renal Physiology, Kone and associates (6) provide evidence that begins to clarify this gap in knowledge and show that Sp1 is one of such positive regulators. Sp1, a promoter-specific factor required for transcription of the SV40 early and late promoters, was the first cloned mammalian transcription factor (2). Sp1 clearly has functional importance, since gene disruption in mice is lethal (3). Sp1, a candidate transcription factor, chosen based on sequence analysis of the α-ENaC promoter, binds to the α-ENaC promoter at a single, specific transcription factor binding site (a cis-element at +222/+229). Thus, Sp1 represents the first described constitutive driver of α-ENaC transcription, which also contributes to maximal aldosterone trans-activation of α-ENaC. Importantly, presented data complement the previous observations of this group. Additional experiments were done to study the role of Sp1 into the context of Dot1a-mediated repression of transcription or aldosterone-mediated induction of α-ENaC transcription. The authors reported that Sp1 acts independently from and overcomes the Dot1a-Af9 repressor complex (Fig. 1). Thus, the model and studies reflect the balance of genetic and epigenetic factors: Sp1 acting on the promoter DNA being the former, and Dot1 acting on histones being the latter. Surprisingly, the authors did not try to place in the global picture regulation of the transcription of ENaC genes by Af17, which competes with Af9 for binding to Dot1a to upregulate transcription of α-ENaC (4) and regulate ENaC-mediated sodium reabsorption and blood pressure (1). Fig. 1. Simplified model for the aldosterone-sensitive regulation of α-epithelial Na+ channel (ENaC) expression. Af9 directly binds +78/+92 of the α-ENaC promoter and recruits Dot1a to this position to basally repress α-ENaC transcription. ... This manuscript presents direct evidence of regulation of α-ENaC by Sp1 and highlights the need for further investigation of mechanisms that initiate or repress transcription of genes encoding ENaC subunits. A few questions are raised from the current study. First, this study is focused on Sp1, but it cannot exclude the possibility that other related factors also interact with the GAGGGCGT boxes of the α-ENaC promoter. Sp1 is part of a large family of proteins, including other Sp factors and the extensive set of Kruppel-like factors. Some of these factors are expressed and functionally important in the cardiorenal system. These factors might have a greater influence than Sp1, either as coactivators, or dominant-negative competitors with Sp1, like Af17, which competes with AF9 for binding to Dot1a (4). Similarly, it is not clear whether Sp1 plays a unique role in regulation of maximal aldosterone-dependent trans-activation of α-ENaC and how other factors like SGK1 modulate this regulation. Furthermore, current studies are performed in cultured mouse inner medullary collecting duct 3 cells in vitro. It would be nice if these findings will be further confirmed in vivo, similar to a recent study where the mice with connecting tubule/cortical collecting duct-specific inactivation of Dot1a revealed greater α-ENaC mRNA levels compared with control (10). Moreover, the functional correlation would improve the physiological significance of these molecular studies. Additional experiments are required to determine whether the Sp1 is functionally important in regulating ENaC or not by overexpressing or knocking down Sp1 and measuring a channel's activity. In conclusion, this study by Yu et al. (6) implicates a key role for the transcription factor Sp1 in regulation of ENaC. The authors propose a mechanism whereby endogenous Sp1 factor is functionally involved in conferring constitutive expression and aldosterone regulation of α-ENaC gene. Sp1 has the potential to be a critical point for understanding the physiological control of ENaC gene expression in the kidney. Importantly, this study is one of the first steps in understanding of genetic and epigenetic regulation of ENaC and emphasizes the need for further investigation of this important area of research.

Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs

At the Drosophila melanogaster bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. Here we demonstrate that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus we discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity.

MRNA levels in control rat liver display strain-specific, hereditary, and AHR-dependent components.

Rat is a major model organism in toxicogenomics and pharmacogenomics. Hepatic mRNA profiles after treatment with xenobiotic chemicals are used to predict and understand drug toxicity and mechanisms. Surprisingly, neither inter- and intra-strain variability of mRNA abundances in control rats nor the heritability of rat mRNA abundances yet been established. We address these issues by studying five populations: the popular Sprague-Dawley strain, sub-strains of Long-Evans and Wistar rats, and two lines derived from crosses between the Long-Evans and Wistar sub-strains. Using three independent techniques – variance analysis, linear modelling, and unsupervised pattern recognition – we characterize extensive intra- and inter-strain variability in mRNA levels. We find that both sources of variability are non-random and are enriched for specific functional groups. Specific transcription-factor binding-sites are enriched in their promoter regions and these genes occur in “islands” scattered throughout the rat genome. Using the two lines generated by crossbreeding we tested heritability of hepatic mRNA levels: the majority of rat genes appear to exhibit directional genetics, with only a few interacting loci. Finally, a comparison of inter-strain heterogeneity between mouse and rat orthologs shows more heterogeneity in rats than mice; thus rat and mouse heterogeneity are uncorrelated. Our results establish that control hepatic mRNA levels are relatively homogeneous within rat strains but highly variable between strains. This variability may be related to increased activity of specific transcription-factors and has clear functional consequences. Future studies may take advantage of this phenomenon by surveying panels of rat strains.

Statistics of protein-DNA binding and the total number of binding sites for a transcription factor in the mammalian genome

BackgroundTranscription factor (TF)-DNA binding loci are explored by analyzing massive datasets generated with application of Chromatin Immuno-Precipitation (ChIP)-based high-throughput sequencing technologies. These datasets suffer from a bias in the information about binding loci availability, sample incompleteness and diverse sources of technical and biological noises. Therefore adequate mathematical models of ChIP-based high-throughput assay(s) and statistical tools are required for a robust identification of specific and reliable TF binding sites (TFBS), a precise characterization of TFBS avidity distribution and a plausible estimation the total number of specific TFBS for a given TF in the genome for a given cell type.ResultsWe developed an exploratory mixture probabilistic model for a specific and non-specific transcription factor-DNA (TF-DNA) binding. Within ChiP-seq data sets, the statistics of specific and non-specific DNA-protein binding is defined by a mixture of sample size-dependent skewed functions described by Kolmogorov-Waring (K-W) function (Kuznetsov, 2003) and exponential function, respectively. Using available Chip-seq data for eleven TFs, essential for self-maintenance and differentiation of mouse embryonic stem cells (SC) (Nanog, Oct4, sox2, KLf4, STAT3, E2F1, Tcfcp211, ZFX, n-Myc, c-Myc and Essrb) reported in Chen et al (2008), we estimated (i) the specificity and the sensitivity of the ChiP-seq binding assays and (ii) the number of specific but not identified in the current experiments binding sites (BSs) in the genome of mouse embryonic stem cells. Motif finding analysis applied to the identified c-Myc TFBSs supports our results and allowed us to predict many novel c-Myc target genes.ConclusionWe provide a novel methodology of estimating the specificity and the sensitivity of TF-DNA binding in massively paralleled ChIP sequencing (ChIP-seq) binding assay. Goodness-of fit analysis of K-W functions suggests that a large fraction of low- and moderate- avidity TFBSs cannot be identified by the ChIP-based methods. Thus the task to identify the binding sensitivity of a TF cannot be technically resolved yet by current ChIP-seq, compared to former experimental techniques. Considering our improvement in measuring the sensitivity and the specificity of the TFs obtained from the ChIP-seq data, the models of transcriptional regulatory networks in embryonic cells and other cell types derived from the given ChIp-seq data should be carefully revised.

Structural property of regulatory elements in human promoters

The capacity of transcription factors to activate gene expression is encoded in the promoter sequences, which are composed of short regulatory motifs that function as transcription factor binding sites (TFBSs) for specific proteins. To the best of our knowledge, the structural property of TFBSs that controls transcription is still poorly understood. Rigidity is one of the important structural properties of DNA, and plays an important role in guiding DNA-binding proteins to the target sites efficiently. After analyzing the rigidity of 2897 TFBSs in 1871 human promoters, we show that TFBSs are generally more flexible than other genomic regions such as exons, introns, 3' untranslated regions, and TFBS-poor promoter regions. Furthermore, we find that the density of TFBSs is consistent with the average rigidity profile of human promoters upstream of the transcription start site, which implies that TFBSs directly influence the promoter structure. We also examine the local rigid regions probably caused by specific TFBSs such as the DNA sequence TATA(A/T)A(A/T) box, which may inhibit nucleosomes and thereby facilitate the access of transcription factors bound nearby. Our results suggest that the structural property of TFBSs accounts for the promoter structure as well as promoter activity.

Predicted transcription factor binding sites as predictors of operons in Escherichia coli and Streptomyces coelicolor.

BackgroundAs a polycistronic transcriptional unit of one or more adjacent genes, operons play a key role in regulation and function in prokaryotic biology, and a better understanding of how they are constituted and controlled is needed. Recent efforts have attempted to predict operonic status in sequenced genomes using a variety of techniques and data sources. To date, non-homology based operon prediction strategies have mainly used predicted promoters and terminators present at the extremities of transcriptional unit as predictors, with reasonable success. However, transcription factor binding sites (TFBSs), typically found upstream of the first gene in an operon, have not yet been evaluated.ResultsHere we apply a method originally developed for the prediction of TFBSs in Escherichia coli that minimises the need for prior knowledge and tests its ability to predict operons in E. coli and the 'more complex', pharmaceutically important, Streptomyces coelicolor. We demonstrate that through building genome specific TFBS position-specific-weight-matrices (PSWMs) it is possible to predict operons in E. coli and S. coelicolor with 83% and 93% accuracy respectively, using only TFBS as delimiters of operons. Additionally, the 'palindromicity' of TFBS footprint data of E. coli is characterised.ConclusionTFBS are proposed as novel independent features for use in prokaryotic operon prediction (whether alone or as part of a set of features) given their efficacy as operon predictors in E. coli and S. coelicolor. We also show that TFBS footprint data in E. coli generally contains inverted repeats with significantly (p < 0.05) greater palindromicity than random sequences. Consequently, the palindromicity of putative TFBSs predicted can also enhance operon predictions.

Some statistical properties of regulatory DNA sequences, and their use in predicting regulatory regions in the Drosophila genome: the fluffy-tail test

BackgroundThis paper addresses the problem of recognising DNA cis-regulatory modules which are located far from genes. Experimental procedures for this are slow and costly, and computational methods are hard, because they lack positional information.ResultsWe present a novel statistical method, the "fluffy-tail test", to recognise regulatory DNA. We exploit one of the basic informational properties of regulatory DNA: abundance of over-represented transcription factor binding site (TFBS) motifs, although we do not look for specific TFBS motifs, per se . Though overrepresentation of TFBS motifs in regulatory DNA has been intensively exploited by many algorithms, it is still a difficult problem to distinguish regulatory from other genomic DNA.ConclusionWe show that, in the data used, our method is able to distinguish cis-regulatory modules by exploiting statistical differences between the probability distributions of similar words in regulatory and other DNA. The potential application of our method includes annotation of new genomic sequences and motif discovery.

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.